This R21 proposal is submitted in response to PA-03-058 Exploratory/Developmental Bioengineering Researcy Grants. Our overall objective is to engineer a novel anti-tumor therapy with eukaryotic expression plasmids that encode therapeutic genes delivered in nonpathogenic mycobacteria, as a safer alternative to viral gene vectors. In particular, we will study the efficacy of wild type Mycobacterium smegmatis, and Mycobacterium bovis BCG, or alternative modified mycobacteria, for the delivery of genes that express functional cytokines or co-stimulatory molecules to bladder tumor cells. Intravesicular BCG therapy, the only US FDA-approved antitumor microbial agent in the US, has contributed to a > 20% decline in death rates from bladder cancer since 1980. Intravesicular BCG augments local production of immune mediators of tumor clearance (IFN-gamma, ICAM-1 and TNF-alpha) and has direct anti-tumor activity. Nevertheless, BCG has no effect in > 20% of cases. M. smegmatis, a species that is less virulent than BCG, inhibits tumor cell growth in vitro more potently than BCG. Previous studies by us, and others, demonstrate that both S. flexneri and S. typhimurium can deliver plasmids to eukaryotic cells for genetic immunization. In a set of pilot experiments, we tested the ability of M. smegmatis to deliver eukaryotic expression plasmids to mammalian cells and clearly demonstrated, for the first time, that M. smegmatis can deliver plasmids expressing the green fluorescent protein from a eukaryotic promoter to macophages. Using this discovery, we plan to develop M. smegmatis and BCG as safe and efficient vectors for the delivery of ICAM-1 and TNF-alpha eukaryotic expression plasmids as gene therapy against bladder carcinoma in humans. Specifically, we propose to: 1. Optimize the ability of wild type M. smegmatis and BCG to deliver Mycobacterial Mammalian Shuttle plasmids (MMSP) to macrophages and murine or human bladder tumor cells in vitro and in mice. 2. Determine whether infection of murine bladder tumor cells with recombinant Mycobacteria harboring MMSP that encode murine ICAM-1 or TNF-alpha augment tumor cell expression of functional ICAM-1 or TNF-alpha in vitro and, 3. Evaluate the ability of recombinant Mycobacteria harboring MMSP that encode ICAM-1 or TNF-alpha genes to enhance tumor regression in a murine MB49 syngeneic orthotopic bladder cancer model using C57BL/6 (immunocompetent).